Introduction

MicroRNAs are a recently discovered class of small, ~19–23-nucleotide non-coding RNA molecules.  They are cleaved from 70–110-nucleotide hairpin precursors and are believed play an important role in translation regulation and degradation of target mRNAs by binding to partially complementary sites in the 3´ untranslated regions (UTRs) of the message (Lim, 2003).  Recent experimental evidence suggests that the number of unique miRNAs in humans could exceed 800, though several groups have hypothesized that there may be up to 20,000 non-coding RNAs that contribute to eukaryotic complexity (Bentwich et al., 2005; Imanishi et al., 2004; Okazaki et al., 2002).
 
Though hundreds of miRNAs have been discovered in a variety of organisms, little is known about their cellular function.  They have been implicated in regulation of developmental timing and pattern formation (Lagos-Quintana et al., 2001), restriction of differentiation potential (Nakahara & Carthew, 2004), regulation of insulin secretion (Stark et al., 2003), and genomic rearrangements (John et al., 2004).
 
Several unique physical attributes of miRNAs - including their small size, lack of poly-adenylated tails, and tendency to bind their mRNA targets with imperfect sequence homology - have made them elusive and challenging to study.  In addition, strong conservation between miRNA family members means that any detection technology must be able to distinguish between ~22-base sequences that differ by only 1–2 nucleotides.  Recent advances in spotted oligonucleotide microarray labeling and detection have enabled the use of this high-throughout technology for miRNA screening.
 
Workflow

The NCode™ suite of miRNA microarray products have been designed and developed for miRNA isolation, labeling, hybridization, and detection.  The figure below shows the workflow of the the NCode™ miRNA labeling system:

 

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Materials

Shipping and Storage

NCode™ Multi-Species miRNA Microarrays are shipped in desiccated, resealable plastic storage containers at room temperature.  Upon receipt, store arrays in the storage container at room temperature protected from light.
 
Contents

Each box contains five microarrays, blocked and ready to use.
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Ordering Information

Sku Name Size Price Qty
15557044 UltraPure™ SSC, 20X 1 L USD 39.00
15553027 UltraPure™ SDS Solution, 10% 4 x 100 mL USD 62.00

Tagged miRNA Hybridization

After you have labeled and purified your miRNA sample (see the miRNA Analysis Labeling and Detection protocol) you are ready to hybridize the purified, tagged miRNA to the microarray.
 
You will need the hybridization components of the NCode™ miRNA Labeling System, available separately.
 
Note on Hybridization Temperature

We have found that increasing the recommended hybridization temperature of 52ºC by a few degrees (to 54–56ºC) will increase hybridization specificity but reduce signal (sensitivity).  We have found that 52ºC provides an optimal balance of sensitivity and specificity with the NCode™ Multi-Species miRNA Microarray.
 
Make sure that the thermometer on your hybridization incubator is properly calibrated.
 
  • NCode™ miRNA microarray products are designed for coverslip hybridizations with volumes of 80 µl or less.
  • Always wear powder-free latex gloves when handling microarrays and coverslips.  Avoid contact with the printed array surface.  The array surface should remain as lint-free and dust-free as possible. Open the slide container just prior to use, and close immediately to store unused slides.
  • The NCode™ Multi-Species miRNA Microarray is printed on the same side of the slide as the barcode.

Before Starting

The following items are supplied in the NCode™ miRNA Labeling Module:

  • 2X Hybridization Solution
  • DEPC-treated water

The following items are supplied by the user:

  • Hybridization chamber (e.g., Corning® Hybridization Chamber, catalog # 2551 or # 40080)
  • Lint-free laboratory wipes
  • Raised-edge coverslips (for the NCode™ Microarray, we recommend LifterSlips™, Erie Scientific catalog # 25x60I-2-4789)
  • Incubator or water bath at 70ºC
  • Incubator or water bath at 75–80ºC
  • Incubator at 52ºC
  • Vortex mixer
  • Microcentrifuge

Tagged miRNA Hybridization Procedure

Use the following procedure to hybridize the purified, tagged miRNA to the NCode™ Multi-Species miRNA Microarray.  This protocol may also be used with anyepoxy-coated glass slide printed with miRNA probes:
 
  1. Thaw the 2X Hybridization Solution by heating it at 70ºC for 10 minutes, and then vortexing to resuspend evenly.  If necessary, repeat heating and vortexing until the buffer is fully resuspended.

  2. The amount of Hybridization Mix per array depends on the coverslip size.  Prepare as follows:


  3. Amount per Array  
    Component 24 × 50 Coverslip 24 × 60 Coverslip
    Tagged miRNA (from Purification Procedure)20 µl20 µl
    DEPC-treated water--5 µl
    2X Hybridization Solution 20 µl25 µl
    Final Volume40 µl50 µl
     
  4. Gently vortex and briefly centrifuge the Hybridization Mix, then incubate the mix at 75–80ºC for 10 minutes.  Hold the mix at the hybridization temperature (52ºC) until loading the array.

  5. Using powder-free latex gloves, inspect the coverslip to ensure it is clean.  If necessary, gently wipe clean with a lint-free laboratory wipe.

  6. Place the slide with the array facing up in an open, clean, dry hybridization chamber.  The array on the NCode™ slide is printed on the same side as the barcode.  The NCode™ microarray comes blocked and ready to use.

  7. Gently vortex and briefly centrifuge the Hybridization Mix.  Then:

    • For LifterSlips™ you may first place the slip on the array with the dull side of the white strips facing down along the length of the slide.  Then position your pipette tip along an open (short) edge of the LifterSlip™, and slowly and carefully pipet the volume of prepared Hybridization Mix from Step 2 under the LifterSlip™, until the array surface underneath is completely covered with the mix.  When pipetting, be careful not to form bubbles under the slip.  If bubbles appear, you may try to remove them by gently tapping the LifterSlip™ with a pipette tip.
    • Alternatively, for either LifterSlips™ or non-raised-edge coverslips, pipet the volume of prepared Hybridization Mix down the center of the array and then carefully apply the coverslip.  Be careful not to form bubbles under the slip.  If bubbles appear, you may try to remove them by gently tapping the coverslip with a pipette tip.


  8. Add the appropriate amount of DEPC-treated water or 2X Hybridization Solution to the hybridization chamber to maintain humidity, and seal the chamber.  Maintaining controlled humidity during hybridization is crucial for successful microarray experiments to prevent the slide from drying out.

  9. Place the hybridization chamber in an incubator at 52°C, and incubate overnight (16–20 hours). During incubation, you may begin preparing Wash Solution 1 and prewarming it to 50ºC.  
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Washing the Array

Following overnight hybridization of the tagged miRNA, perform the array wash procedure in this section prior to hybridization of the Alexa Fluor® Capture Reagents.
 
Before Starting

The following items are supplied by the user:

  • Wash Solution 1: 2X SSC/0.2% SDS (may be prepared from UltraPure™ 20X SSC and UltraPure™ 10% SDS Solution, available from Invitrogen)
  • Wash Solution 2: 2X SSC
  • Wash Solution 3: 0.2X SSC
  • Clean slide rack
  • 3 clean wash containers, capable of completely submerging an array slide in a slide rack (e.g., 400 ml).
  • An additional wash container or squirt bottle for washing off the coverslip
  • Incubator or water bath at 50ºC
  • Vortex mixer
  • Tabletop centrifuge with a microtiter plate rotor adapter capable of holding a slide rack, or with a slide holder


Prepare Wash Solutions

Prepare appropriate volumes of Wash Solution 1 (2X SSC/0.2% SDS), Wash Solution 2 (2X SSC), and Wash Solution 3 (0.2X SSC) prior to beginning the wash procedure. Prepare enough of each solution to fully submerge the slide in a slide rack in a wash container filled with the solution.
 
The table below provides volumes for preparing 2 liters of each solution from UltraPure™ 20X SSC, UltraPure™ 10% SDS, and dH20:
 
                                     Wash Solution 1        Wash Solution 2        Wash Solution 3
UltraPure™ 20X SSC             200 ml                           200 ml                            20 ml
UltraPure™ 10% SDS             20 ml                               —                                  —
dH20                                     to 2 liters                      to 2 liters                      to 2 liters
 
The following procedure has been designed for use with epoxy-coated, glass slide microarrays. If you have hybridized the tagged miRNA to a different type of microarray, use solutions and protocols appropriate for that array.  We do not recommend using compressed air to dry the slides following washing, as this can result in streaking and high background.  Always wear powder-free latex gloves when handling arrays.  Avoid contact with the printed array surface. The array surface should remain as lint-free and dust-free as possible.

Array Wash Procedure

Follow the procedure below to wash the array slide hybridized with tagged miRNA from Tagged miRNA Hybridization Procedure, Step 8.
 

  1. Prewarm the prepared volume of Wash Solution 1 to 50ºC. 
  2. Place a slide rack in a wash container, and fill the container with prewarmed Wash Solution 1 until the rack is completely submerged in the wash solution.
  3. To wash the coverslip from the array slide, use one of two methods:
    • Fill another wash container with prewarmed Wash Solution 1.  Remove the array slide from the hybridization chamber and, holding the slide at the barcode end, submerge it in Wash Solution 1.  Gently move it back and forth in solution until the coverslip falls off.
    • Fill a squirt bottle with prewarmed Wash Solution 1.  Remove the array slide from the hybridization chamber
  4. Quickly transfer the slide to the slide rack in prewarmed Wash Solution 1 from Step 2.  Make sure the slide is completely submerged in the solution.  Do not expose the slide to air for more than a few seconds to avoid air-drying.
  5. Wash the array in Wash Solution 1 for 10–15 minutes at room temperature with gentle agitation (150–200 rpm).
  6. Fill a clean wash container with Wash Solution 2 at room temperature.  Transfer the slide rack with the slide to this wash container, and wash with gentle agitation at room temperature for 10–15 minutes.  Do not expose the slide to air for more than a few seconds, and make sure that the slide is completely submerged in Wash Solution 2.
  7. Fill a clean wash container with Wash Solution 3 at room temperature.  Transfer the slide rack with the slide to this wash container, and wash with gentle agitation at room temperature for 10–15 minutes.  Do not expose the slide to air for more than a few seconds, and make sure that the slide is completely submerged in Wash Solution 3.
     
    Important:  Perform the next step quickly.  To avoid high background on the array, do not allow the array surface to air dry prior to centrifugation.
  8. Prepare a centrifuge with a microtiter plate rotor adapter that will accept the slide rack containing the array slide. (Balance the opposing arm of the rotor with a slide rack containing an equivalent number of empty slides.)  Quickly transfer the slide rack with the slide to the centrifuge, and immediately spin for 2–4 minutes at 600 × g to dry.  Do not centrifuge at higher speeds, or the slide might break.


 
Proceed to Detection with Alexa Fluor® Capture Reagents

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Detection with Alexa Fluor® Capture Reagents

Following the array wash, hybridize the Alexa Fluor® Capture Reagents to the tagged miRNA using the procedure in this section.
 
The following procedure has been designed for use with epoxy-coated, glass slide microarrays.  If you are using a different type of array, use hybridization solutions and protocols appropriate for that array.
 
Before Starting

The following items are supplied in the NCode™ miRNA Labeling Module:

  • Alexa Fluor® 3 Capture Reagent and Alexa Fluor® 5 Capture Reagent
  • 2X Hybridization Solution
  • DEPC-treated water

The following items are supplied by the user:

  • Hybridization chamber (e.g., Corning® Hybridization Chamber, catalog # 2551 or # 40080)
  • Lint-free laboratory wipes
  • Raised-edge coverslips (e.g., LifterSlips™, Erie Scientific, catalog # 25x60I-2-4789)
  • Incubator or water bath at 50ºC
  • Incubator or water bath at 70ºC
  • Incubator or water bath at 75–80ºC
  • Incubator at 62ºC
  • Vortex mixer
  • Microcentrifuge

Alexa Fluor® 3 and Alexa Fluor® 5 Capture Reagents

Alexa Fluor® 3 and Alexa Fluor® 5 Capture Reagents* contain branched DNA polymers ("dendrimers”), each with a core that consists of a matrix of double-stranded DNA, and an outer surface comprised of hundreds of singled-stranded “arms.”  The surface arms carry ~900 Alexa Fluor® molecules, as well as highly specific sequences complementary to the ligated tags on the hybridized miRNAs.
 
Note that Alexa Fluor® 3 is identical to Alexa Fluor® 546, and Alexa Fluor® 5 is identical to Alexa Fluor® 647. Excitation and emission maxima are listed below:

                                                                 Excitation                                    Emission
Alexa Fluor™ 3:                                       556 nm                                          573 nm
Alexa Fluor™ 5:                                       650 nm                                          665 nm

*Capture reagents contain 3DNA™ reagent manufactured under license from Genisphere, Inc.

  • During the following procedure, minimize exposure of Alexa Fluor® Capture Reagents and the hybridized array to direct light, to avoid photobleaching.
  • Always wear powder-free latex gloves when handling arrays.  Avoid contact with the printed array surface. The array surface should remain as lint-free and dust-free as possible. 

Preparing the Alexa Fluor® Capture Reagents

Before hybridization, prepare the Alexa Fluor® Capture Reagents as follows:
 
  1. Thaw the Alexa Fluor® 3 and/or Alexa Fluor® 5 Capture Reagents in the dark at room temperature for 20 minutes.

  2. Vortex the tube(s) at maximum speed for 3 seconds, then centrifuge briefly.

  3. Incubate in the dark at 50–55ºC for 10 minutes.

  4. Vortex the tube(s) at maximum speed for 3–4 seconds, and then centrifuge briefly to collect the contents.
 
Hybridization of Alexa Fluor® Capture Reagents Procedure

Follow the protocol below to hybridize the Alexa Fluor® Capture Reagents to the NCode™ Multi-Species miRNA Microarray.  This protocol may also be used with anyepoxy-coated glass slide printed with miRNA probes:
 
  1. Thaw the 2X Hybridization Solution by heating it at 70ºC for 10 minutes, and then vortexing to resuspend evenly.  If necessary, repeat heating and vortexing until the solution is fully resuspended.  When resuspended, centrifuge for 1 minute.

  2. Prepare the Hybridization Mix.  Use Alexa Fluor® Capture Reagents thawed as described above.  The amount of Hybridization Mix per array depends on the coverslip size.
  3.  
    Note:  The following table shows amounts for a dual-color hybridization.  For single-color hybridizations, replace the volume of the second capture reagent with DEPC-treated water:

     Amount per Array
    Component 24 × 50 Coverslip 24 × 60 Coverslip
    Alexa Fluor® 3 Capture Reagent 2.5 µl 2.5 µl
    Alexa Fluor® 5 Capture Reagent 2.5 µl2.5 µl
    DEPC-treated water 15 µl 20 µl
    2X Hybridization Solution20 µl 25 µl
    Final Volume 40 µl 50 µl

     
  4. Gently vortex and briefly centrifuge the Hybridization Mix, then incubate in the dark at 75–80ºC for 10 minutes.  After incubation, hold the mix at the hybridization temperature (62ºC) in the dark until loading the array.

  5. Wearing powder-free latex gloves, inspect the coverslip to ensure it is clean.  If necessary, gently wipe clean with a lint-free laboratory wipe.

  6. Place the washed array slide from face-up in an open hybridization chamber.
     
    Important:   When performing the following steps, to the extent possible shield the array from direct light, to avoid photobleaching of the capture reagents.

  7. Gently vortex and briefly centrifuge the Hybridization Mix.  Then either position a LifterSlip™ on the array and pipet the hybridization mix under the slip, or pipet the hybridization mix onto the array and then apply the coverslip, as previously described in Step 6, Tagged miRNA Hybridization.  Once again, be careful to avoid the formation of bubbles under the coverslip.

  8. Add the appropriate amount of DEPC-treated water or 2X Hybridization Solution to the hybridization chamber to maintain humidity, and seal the chamber.  Maintaining controlled humidity during hybridization is crucial to prevent the slide from drying out.

  9. Place the hybridization chamber in an incubator at 62°C for NCode™ arrays.  Incubate in the dark for 4 hours.  During incubation, you may prepare Wash Solution 1 and begin prewarming it to 60ºC.

 
Proceed directly to Final Array Wash.
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Final Array Wash

Following the 4-hour hybridization of the Alexa Fluor® Capture Reagents, perform the final array wash procedure as described in this section.
 
Before Starting

The following items are supplied by the user:

  • Wash Solution 1: 2X SSC/0.2% SDS (may be prepared from UltraPure™ 20X SSC and UltraPure™ 10% SDS Solution, available from Invitrogen; see page vii) •Wash Solution 2: 2X SSC
  • Wash Solution 3: 0.2X SSC
  • Clean slide rack
  • 3 clean wash containers, capable of completely submerging an array slide in a slide rack (e.g., 400 ml).
  • An additional wash container or squirt bottle for washing off the coverslip
  • Incubator or water bath at 60ºC
  • Vortex mixer
  • Tabletop centrifuge with a microtiter plate rotor adapter capable of holding a slide rack, or with a slide holder

Prepare Wash Solutions

Prepare appropriate volumes of Wash Solution 1 (2X SSC/0.2% SDS), Wash Solution 2 (2X SSC), and Wash Solution 3 (0.2X SSC) prior to beginning the wash procedure.  Prepare enough of each solution to fully submerge the array slide in a slide rack in a wash container filled with the solution.
 
The table below provides volumes for preparing 2 liters of each solution from UltraPure™ 20X SSC, UltraPure™ 10% SDS, and dH20:

  Wash Solution 1 Wash Solution 2 Wash Solution 3
UltraPure™ 20X SSC200 ml 200 ml 20 ml
UltraPure™ 10% SDS 20 ml----
dH20 to 2 liters to 2 liters to 2 liters

Final Array Wash
 
The following procedure has been designed for use with epoxy-coated, glass slide microarrays.  If you have hybridized the tagged miRNA to a different type of microarray, use solutions and protocols appropriate for that array.
  
Always wear powder-free latex gloves when handling arrays. Avoid contact with the printed array surface.  The array surface should remain as lint-free and dust-free as possible.  We do not recommend using compressed air to dry the slides following washing, as this can result in streaking and high background.  Be careful to minimize exposure of the hybridized array to direct light, to avoid photobleaching.
 

Follow the procedure below to wash the array slide from Detection with Alexa Fluor® Capture Reagents, Step 8.
 
  1. Prewarm the prepared volume of Wash Solution 1 to 60°C.

  2. Place a slide rack in a wash container, and fill the container with prewarmed Wash Solution 1 until the rack is completely submerged in the wash solution.

  3. To wash the coverslip from the array slide, use one of two methods:

    • Fill another wash container with prewarmed Wash Solution 1.  Remove the array slide from the hybridization chamber and, holding the slide at the barcode end, submerge it in Wash Solution 1.  Gently move it back and forth in solution until the coverslip falls off.
    • Fill a squirt bottle with prewarmed Wash Solution 1.  Remove the array slide from the hybridization chamber and, holding the slide at a downward angle, gently squirt the wash solution onto the surface until the coverslip falls off.


  4. Quickly transfer the slide to the slide rack in prewarmed Wash Solution 1 from Step 2.  Make sure the slide is completely submerged in the solution.  Do not expose the slide to air for more than a few seconds to avoid air-drying.

  5. Wash the array in Wash Solution 1 for 10–15 minutes at room temperature with gentle agitation (150–200 rpm).

  6. Fill a clean wash container with Wash Solution 2 at room temperature.  Transfer the slide rack with the slide to this wash container, and wash with gentle agitation at room temperature for 10–15 minutes.  Do not expose the slide to air for more than a few seconds, and make sure that the slide is completely submerged in Wash Solution 2.

  7. Fill a clean wash container with Wash Solution 3 at room temperature.  Transfer the slide rack with the slide to this wash container, and wash with gentle agitation at room temperature for 10–15 minutes.  Do not expose the slide to air for more than a few seconds, and make sure that the slide is completely submerged in Wash Solution 3.
     
    Important:
       Perform the next step quickly.  To avoid high background on the array, do not allow the array surface to air dry prior to centrifugation.

  8. Prepare a centrifuge with a microtiter plate rotor adapter that will accept the slide rack containing the array slide. (Balance the opposing arm of the rotor with an slide rack containing an equivalent number of empty slides.)  Quickly transfer the slide rack with the slide to the centrifuge, and immediately spin for 2–4 minutes at 600 × g to dry.  Do not centrifuge at higher speeds, or the slide might break.

Keep the arrays in the dark, protected from light, until scanning.  We recommend scanning the array within ½ hour after the final wash step, to avoid photobleaching.  See Array Imaging and Analysis.
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Array Imaging and Analysis

Following the final wash step, arrays should be scanned immediately to minimize photobleaching of the Alexa Fluor® dyes.
 
Scanning the Microarray

  • The array should be shielded from direct light and scanned within ½ hour of completion the final wash, to minimize photobleaching.
  • The microarray slide may be scanned using a standard digital microarray scanner.  We recommend a scanner with a bit depth of at least 16 bits/pixel.  The GenePix® 4000B (Molecular Devices) is a common microarray scanner, and includes software for analyzing the scanned image.
  • Alexa Fluor® 3 and 5 have excitation and emission maxima identical to Alexa Fluor® 546 and 647, respectively. Program your scanner accordingly.


                                                               Excitation                             Emission
Alexa Fluor® 3:                                       556 nm                                  573 nm
Alexa Fluor® 5:                                       650 nm                                  665 nm
 

  • Follow the instructions provided with your scanner for adjusting the photomultiplier tube (PMT) settings.  It is important to adjust the PMT setting for each channel for maximum dynamic range and channel balance.
  • A typical lower limit of detection (LLD) is 8 times the median local background of all array features.  The signal/background (S/B) ratio is calculated by dividing the median signal of positive features by the median background.
  • Be careful to position the slide in the proper orientation in the microarray scanner.  If no signal is apparent after scanning, double-check the orientation of the slide.  Consult your scanner documentation for details.


When scanning dual-color arrays, we recommend examining the image histogram (available with GenePix® Pro software) to determine whether the signal intensities in the two channels are comparable.

GAL Files

Many arrays, including the NCode™ Multi-Species miRNA Microarray, provide a GAL (GenePix® Array List) file that includes the positions, names, and probe ID’s of all probes on the array, in tabular format.  Most major microarray software applications, including GenePix® Pro and ScanArray®, can combine the tab-delimited information from the GAL file with the spot intensity data from the scanned array to generate a results file with all the data for the experiment.
 
Note:   If your software does not accept files in GAL format, open the file in a spreadsheet program like Microsoft® Excel and reformat the information in a compatible configuration, or manually correlate information with your spot intensity data. Consult your microarray analysis software for details.
 
Downloading the NCode™ Multi-Species miRNA Microarray GAL File

Go to the NCode™ website at www.lifetechnologies.com/ncode and navigate to the download page.  GAL files are listed by microarray lot number.  The lot number is printed on each box of NCode™ microarray slides.  The same lot number applies to all the slides in the box.
 
Select the GAL file your box of slides, and download it to a location on your computer.  Then follow the instructions provided with your microarray analysis software to combine the GAL file information with your scan data.
 

Troubleshooting

Problem
Cause
Solution
Coverslip stuck to array surface
Hybridization chamber not properly sealed or humidified
Make sure that the hybridization chamber is properly sealed with the correct amount of liquid prior to incubation of the hybridized array
 
Inadequate volume of hybridization buffer used for coverslip size
Make sure that the hybridization buffer completely covers the array surface under the coverslip.
Low or no overall fluorescent signal intensity on the array
Photobleaching of the fluorescent labels
Avoid direct exposure of the hybridized array and the mixes containing the fluorescent dyes to light. Perform all hybridization and wash procedures in low light conditions.
 
Incubation temperatures during hybridization were incorrect
Incubation temperatures that are too high will result in lower signal. Check the temperatures of all incubators with a calibrated thermometer.
 
Degraded starting material
Use isolated small RNA as starting material for labeling and hybridization, and follow appropriate guidelines for handling RNA to prevent RNase contamination. Always use fresh samples or samples frozen at -80 °C. If you are starting with total RNA, analyze it by agarose/ethidium bromide gel electrophoresis prior to isolation of small RNA.
 
Array slide scanned in wrong orientation
Check the position of the slide in the scanner; reposition and rescan if necessary
High or uneven background on the array
Wash solution residue on the slide
Transfer the slide quickly between wash steps, and centrifuge immediately after the final wash step to quickly dry the slide. Avoid exposing the slide to air between washes for more than a few seconds. Improperly dried wash solution will appear as streaks on the slide.
 
Dehydration of the hybridization buffer
This frequently appears as high background around the edges of the array coverslip. Make sure that the hybridization buffer completely covers the array surface under the coverslip, and that humidity is maintained during incubation.
 
Improper array handling
Always wear powder-free gloves when handling the array, and avoid touching the slide surface.
 
Scanner laser and/or PMT settings are too high
Increasing these settings to adjust for low signal will increase array background. If the fluorescent signal is too low, see above.
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References

  1. Bentwich, I., Avniel, A., Karov, Y., Aharonov, R., Gilad, S., Barad, O., Barzilai, A., Einat, P., Einav, U., Meiri, E., Sharon, E., Spector, Y., and Bentwich, Z. (2005) Identification of hundreds of conserved and nonconserved human microRNAs. Nature Genet., 37, 766-770

  2. Goff, L. A., Yang, M., Bowers, J., Getts, R. C., Padgett, R. W., and Hart, R. P. (2005) Rational probe optimization and enhanced detection strategy for microRNAs using microarrays. RNA Biology, 2, published online

  3. Imanishi, T., Itoh, T., Suzuki, Y., and O'Donovan, C. (2004) Integrative annotation of 21,037 human genes validated by full-length cDNA clones. PLoS Biol., 2, e162

  4. John, B., Enright, A. J., Aravin, A., Tuschl, T., Sander, C., and Marks, D. S. (2004) Human MicroRNA Targets. PLoS Biol., 2, e363

  5. Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001) Identification of novel genes coding for small expressed RNAs. Science, 294, 853-858

  6. Lim, L. P., Glasner, M. E., Yekta, S., Burge, C. B., Bartel,D. P. (2003) Vertebrate microRNA Genes. Science, 299, 1540

  7. Nakahara, K., and Carthew, R. W. (2004) Expanding roles for miRNAs and siRNAs in cell regulation. Curr Opin Cell Biol, 16, 127-133

  8. Okazaki, Y., Furuno, M., Kasukawa, T., and Adachi, J. (2002) Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature, 420, 563-573

  9. Stark, A., Brennecke, J., Russell, R. B., and Cohen, S. M. (2003) Identification of Drosophila MicroRNA Targets. PLoS Biol., 1, E60.

  10. Xie, X., Lu, J., Kulbokas, E. J., Golub, T. R., Mootha, V., Lindblad-Toh, K., Lander, E. S., and Kellis, M. (2005) Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals. Nature, 434, 338-345

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